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Carlo Knupp

Publications and source records attributed to Carlo Knupp.

16 recordsLinked to original sources
PubMed2006

The myosin filament superlattice in the flight muscles of flies: A-band lattice optimisation for stretch-activation?

Low-angle X-ray diffraction patterns from relaxed fruitfly (Drosophila) flight muscle recorded on the BioCat beamline at the Argonne Advanced Photon Source (APS) show many features similar to such patterns from the "classic" insect flight muscle in Lethocerus, the giant water bug, but there is a characteristically different pattern of sampling of the myosin filament layer-lines, which indicates the presence of a superlattice of myosin filaments in the Drosophila A-band. We show from analysis of the structure factor for this lattice that the sampling pattern is exactly as expected if adjacent four-stranded myosin filaments, of repeat 116 nm, are axially shifted in the hexagonal A-band lattice by one-third of the 14.5 nm axial spacing between crowns of myosin heads. In addition, electron micrographs of Drosophila and other flies (e.g. the house fly (Musca) and the flesh fly (Sarcophaga)) combined with image processing confirm that the same A-band superlattice occurs in all of these flies; it may be a general property of the Diptera. The different A-band organisation in flies compared with Lethocerus, which operates at a much lower wing beat frequency (approximately 30 Hz) and requires a warm-up period, may be a way of optimising the myosin and actin filament geometry needed both for stretch activation at the higher wing beat frequencies (50 Hz to 1000 Hz) of flies and their need for a rapid escape response.

Actin Cytoskeleton↗
PubMed2006

Structural correlation between collagen VI microfibrils and collagen VI banded aggregates.

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.

Aged↗
PubMed2006

Reprint of "Structural correlation between collagen VI microfibrils and collagen VI banded aggregates" [J. Struct. Biol. 154 (2006) 312-326].

Collagen VI is a component of the extracellular matrix that is able to form structural links with cells. Collagen VI monomers cross-link into tetramers that come together to form long molecular chains known as microfibrils. Collagen VI tetramers are also the most likely candidates for the formation of banded aggregates with an axial periodicity of about 105 nm that are seen in the retinas of people suffering from age-related macular degeneration and Sorsby's fundus dystrophy, in the vitreous of patients with full thickness macular holes and in the intervertebral discs of normal individuals. Here, a protocol is developed to carry out a structural comparison between the microfibrils, which are known to be made of collagen VI tetramers, and the banded aggregates. The comparison shows that the banded aggregates are easily explained as being a lateral assembly of microfibrils, thus supporting the hypothesis that they too are made of collagen VI. Understanding the role played by the collagen VI aggregates in normal and pathological conditions will help to throw light on the pathologies with which they are associated.

Journal Article↗
PubMed2005

Molecular packing in network-forming collagens.

Different collagen types can vary considerably in length, molecular weight, chemical composition, and the way they interact with each other to form molecular aggregates. Collagen Types IV, VI, VIII, X, and dogfish egg case collagen make linear and lateral associations to form open networks rather than fibers. The roles played by these network-forming collagens are diverse: they can act as support and anchorage for cells and tissues, serve as molecular filters, and even provide protective permeable barriers for developing embryos. Their functional properties are intimately linked to their molecular organization. This Chapter reviews what is known about the molecular structure of this group of collagens, describes the ways the molecules interact to form networks, and-despite the large variations in molecular size-identifies common aggregation themes.

Chemical Phenomena↗
PubMed2004

New X-ray diffraction observations on vertebrate muscle: organisation of C-protein (MyBP-C) and troponin and evidence for unknown structures in the vertebrate A-band.

Previous low-angle X-ray diffraction studies of various vertebrate skeletal muscles have shown the presence of two rich layer-line patterns, one from the myosin heads and based on a 429 A axial repeat, and one from actin filaments and based on a repeat of about 360-370 A. In addition, meridional intensities have been seen from C-protein (MyBP-C; at about 440 A and its higher orders) and troponin (at about 385 A and its orders). Using preparations of intact, relaxed, bony fish fin muscles and the ID-02 low-angle X-ray camera at the ESRF with a 10 m camera length we have now seen numerous, hitherto unreported, sampled, X-ray layer-lines many of which do not fit onto the previously observed repeats and which require interpretation. The new reflections all fall on the normal ("vertical") hexagonal lattice row-lines in the highly sampled, almost "crystalline", low-angle diffraction X-ray patterns from bony fish muscle, indicating that they all arise from the muscle A-band. However, they do not fall on a single axial repeat. In direct confirmation of our previous analysis, some of these new reflections are explained by the interaction in resting muscle between the N-terminal ends of myosin-bound C-protein molecules with adjacent actin filaments, possibly through the Pro-Ala-rich region. Other newly observed reflections lie on a much longer repeat, but they are most easily interpreted in terms of the arrangement of troponin on the actin filaments. If this is so, then the implication is that the actin filaments and their troponin complexes are systematically arranged in the fish muscle A-band lattice relative to the myosin head positions, and that these newly observed X-ray reflections, when fully analysed, will report on the shape and distribution of troponin molecules in the resting muscle A-band. The less certain contributions of titin and nebulin to these new reflections have also been tested and are described. Many of the new reflections do not appear to come from these known structures. There must be structural features of the A-band that have not yet been described.

Animals↗
PubMed2004

MusLABEL: a program to model striated muscle A-band lattices, to explore crossbridge interaction geometries and to simulate muscle diffraction patterns.

The program MusLABEL has been devised as a simple aid both in understanding the origin and appearance of fibre diffraction patterns from helical structures and also to simulate the structure and some features of the diffraction patterns from striated muscles and their filament components. Helices are common as preferred conformations in both natural and synthetic macromolecules (e.g. DNA, alpha -helices, polysaccharides, synthetic polymers), and they also occur frequently in extended macromolecular aggregates (e.g. actin filaments, myosin filaments, microtubules, amyloid filaments etc). For this reason, a simple way of visualising the kinds of diffraction patterns that these filament structures can give, particularly for the actin and myosin filaments in muscle, can have educational value and can also be useful as a quick means of evaluating possible symmetries in structural interpretations of diffraction data before embarking on full helical diffraction analysis. A feature of the MusLABEL program is that, when a particular kind of A-band lattice has been set up, for example for vertebrate striated muscle or insect flight muscle, additional parameters can be defined both to describe the limits to the azimuthal and axial ranges over which a myosin head can search for an actin binding site and also to describe the size and position of an actin 'target area' assuming that the azimuthal position of an actin monomer has a large effect in determining whether or not a myosin head can bind to it. By this means the effects of lattice geometry on head attachment can be explored and the diffraction effects of specific labelling patterns on actin can be calculated and simulated. The MusLABEL program, running under Microsoft Windows, is available free on the CCP13 website (www.ccp13.ac.uk) where further documentation is given.

Animals↗
PubMed2004

Modulation of Sub-RPE deposits in vitro: a potential model for age-related macular degeneration.

PURPOSE: Sub-RPE deposits form in a variety of conditions most notably in age-related macular degeneration. The purpose of this study was to generate sub-RPE deposits in vitro and to test the hypotheses that high protein concentrations or retinal homogenate increase deposit formation and that a challenge with tumor necrosis factor (TNF)-alpha or metalloproteinase (MMP)-2 decreases such deposits. METHODS: ARPE-19 cells were grown on plastic and on collagen type I-coated membrane inserts in media containing various concentrations of fetal calf serum (FCS), bovine serum albumin, or porcine retinal homogenate. In addition, cells grown on membrane inserts were treated with TNF-alpha or MMP-2. Sub-RPE deposits were assessed by electron microscopy and classified into fibrillar, condensed, banded, and membranous subtypes. The area of the micrograph occupied by each type was estimated with a point-counting technique. MMP-2 activity was assessed in tissue culture supernatants by zymography. RESULTS: With increasing time in culture, total deposit formation did not change, but the amount of condensed material deposited by ARPE-19 cells increased while the fibrillar component decreased. Albumin challenge resulted in an increased amount of deposit, predominantly of the membranous type. Challenge with retinal homogenate led to a greater net deposit formation with significant increases in the condensed and banded forms. Cells treated with TNF-alpha or MMP-2 showed a dramatic reduction in all types of sub-RPE deposit. Zymography demonstrated that unchallenged cells produced predominantly MMP-2. Retinal homogenate challenge reduced the total amount of active MMP-2 produced, and TNF-alpha stimulated MMP-9 production. CONCLUSIONS: Sub-RPE deposits formed in vitro share ultrastructural features with those seen in vivo. Deposit formation can be modulated by challenge with retinal homogenate, TNF-alpha, or MMP-2. Significantly, the results provide proof of the principle that sub-RPE deposits can be formed and modified in vitro.

Animals↗
PubMed2003

Structural evidence for the interaction of C-protein (MyBP-C) with actin and sequence identification of a possible actin-binding domain.

C-protein (MyBP-C) is a myosin-binding protein that is usually seen in two sets of seven to nine positions in the C-zones in each half of the vertebrate striated muscle A-band. Skeletal muscle C-protein is a modular structure containing ten sub-domains (C1 to C10) of which seven are immunoglobulin-type domains and three (C6, C7 and C9) are fibronectin-like domains. Cardiac muscle C-protein has an extra N-terminal domain (C0) and also some sequence insertions, one of which provides phosphorylation sites. It is conceivable that C-protein has both a structural and regulatory role within the sarcomere. The precise mode of binding of C-protein to the myosin filament has not been determined. However, detailed ultrastructural studies have suggested that C-protein, which binds to myosin, can give rise to a longer periodicity (about 435A) than the intrinsic myosin filament repeat of 429A. The reason for this has remained a puzzle for over 25 years. Here we show by modelling and computation that the presence of this longer periodicity could be explained if the myosin-binding part of C-protein binds to myosin with the expected 429A repeat, but if there are systematic interactions of the N-terminal end of C-protein with the neighbouring actin filaments in the hexagonal lattice of filaments in the A-band. We also show that if they occur these interactions would probably only arise in defined muscle states. Further analysis of the MyBP-C sequence identifies a possible actin-binding domain in the Pro-Ala-rich sequence found at the N terminus of skeletal MyBP-C and between domains C0 and C1 in the cardiac sequence.

Actins↗
PubMed2003

Molecular packing in network-forming collagens.

Collagen is the most abundant protein among vertebrates and occurs in virtually all multicellular animals. Collagen molecules are classified into 21 different types and differ in their sequence, weight, structure, and function, but they can be broadly subdivided into families. Type IV, VI, VIII, X, and dogfish egg case collagens belong to the network-forming family. Here, we summarise what is known about the way these collagen molecules pack to form networks. In addition the main structural characteristics of the network-forming collagens are compared and discussed.

Animals↗
PubMed2002

Titin organisation and the 3D architecture of the vertebrate-striated muscle I-band.

The giant muscle protein titin (connectin) is known to serve as a cytoskeletal element in muscle sarcomeres. It elastically restrains lengthening sarcomeres, it aids the integrity and central positioning of the A-band in the sarcomere and it may act as a template upon which some sarcomeric components are laid down during myogenesis. A puzzle has been how titin molecules, arranged systematically within the hexagonal A-band lattice of myosin filaments, can redistribute through the I-band to their anchoring sites in the tetragonal Z-band lattice. Recent work by Liversage and colleagues has suggested that there are six titin molecules per half myosin filament. Since there are two actin filaments per half myosin filament in a half sarcomere, this means that there are three titin molecules interacting with each Z-band unit cell containing one actin filament in the same sarcomere and one of opposite polarity from the next sarcomere. Liversage et al. suggested that the three titins might be distributed with two on an actin filament of one polarity and one on the filament of opposite polarity. Here, we build on this suggestion and discuss the transition of titin from the A-band to the Z-band. We show that there are good structural and mechanical reasons why titin might be organised as Liversage et al., suggested and we discuss the possible relationships between A-band arrangements in successive sarcomeres along a myofibril.

Animals↗
PubMed2002

Collagen VI assemblies in age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of incurable blindness in the developed world. Little is known about the pathogenesis of this condition, but deposits in Bruch's membrane and immediately beneath the retinal pigment epithelium are frequent findings associated with this disease. Within these deposits, molecular assemblies with an approximately 100-nm axial periodicity are seen. Two types of assembly are present: one exhibiting transverse double bands of protein density that are 30nm apart and repeat axially every approximately 100nm; the other with transverse double bands of protein density, 30nm apart and repeating axially every approximately 50nm. In this second type of assembly, more prominent pairs of bands alternate with less prominent ones. By comparison with analogous aggregates found in the vitreous of a patient with a full-thickness macular hole, collagen VI was singled out as the most probable protein constituent of the AMD aggregates. Possible models for the aggregation patterns of these assemblies are discussed in terms of collagen VI dimers and tetramers. Understanding the structure and chemical composition of the assemblies within the AMD basal deposits may prove of great help in understanding the pathophysiology of AMD itself.

Aging↗
PubMedDate not supplied

Analysis of the collagen VI assemblies associated with Sorsby's fundus dystrophy.

Age-related macular degeneration is the leading cause of blindness in the Western world, and the pathophysiology of the condition is largely unknown. However, it shares many clinical and pathological features with Sorsby's fundus dystrophy (SFD), an autosomal dominant disease, known to be associated with mutations in the TIMP-3 gene. In Bruch's membrane of both conditions, there are molecular assemblies with distinct transverse bands occurring with a periodicity of about 100 nm. Similar assemblies were also found in the vitreous of a patient with full-thickness macular holes and were identified as being made of collagen VI. The assemblies found in the eye with SFD can be classified into two types, both with a 105-nm axial repeat, but one showing pairs of narrow bands about 30 nm apart and the other showing a single broad band in every repeat. By comparison with the assemblies in the vitreous, collagen VI is considered to be the most likely protein in these assemblies. Furthermore, both of the assemblies associated with SFD can be explained in terms of collagen VI tetramers, one in which the tetramers bind to the mutant tissue inhibitor of metalloproteinases-3 (the gene product of TIMP-3) and the other in which little or no binding occurs. TIMP-3 bound to collagen VI may be more resistant to degradation and create an imbalance between the normal amount of TIMP-3 and matrix metalloproteinases (the substrate of TIMPs) in Bruch's membrane with consequent disruption of the normal metabolic processes. Understanding the structure of these collagen VI/TIMP assemblies in Bruch's membrane may prove to be important for understanding the pathophysiology of age-related macular degeneration.

Aged↗